Sodium 4-morpholin-1-ylethylsulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-29 to 2016-09-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-morpholinyl)ethansulfonic acid hydrate

Cas Number:

1266615-59-1

Molecular formula:

C6H13NO4S * xH2O

IUPAC Name:

2-(4-morpholinyl)ethansulfonic acid hydrate

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system
- source of S9 : The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2.; D-35394 Giessen, Germany)
- concentration or volume of S9 mix and S9 in the final culture medium : 10% S9 mix in final culture medium
- quality controls of S9: enzymatic activity, sterility, metabolic capability

Test concentrations with justification for top dose:

First and second experiment: 16, 50, 160, 500, 1600, 5000 µg/plate, where 5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine

Remarks:

without S9 mix, TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix, S. typhimurium strains and E. coli WP2 uvrA

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix, TA100 and TA1535

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix, TA1537

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix, E.coli WP2 uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: number of revertant colonies (with TA98 and TA100)

Evaluation criteria:

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
The pH of the test item stock solution (prepared for the highest concentration) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at the highest concentration of 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the different overlays without test item stock solution was in the range of 7.12 - 7.30, and the pH of overlays completed with test item stock solution was ~7 in all cases.
Extremes of pH could have influencing effect on the mutagenicity results; however in this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the result interpretation.
- Water solubility: 50 mg/mL
- Precipitation and time of the determination: no

RANGE-FINDING/SCREENING STUDIES:
Based on the solubility test, a stock solution with a concentration of 50 mg/mL was prepared in ultrapure water and diluted in 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 μg/plate of the test item.
The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.

STUDY RESULTS
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
Sporadically increased revertant colony numbers were noticed in the performed experiments; these increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the Initial Mutation Test (Plate Incorporation Test) in S. typhimurium TA1537 strain at 16 μg/plate, in presence of metabolic activation (+S9). This higher value however remained in the range of the corresponding vehicle historical control data. The mutation rate was 1.81, which was far below the genotoxicological threshold for being positive, was a unique value without any biological significance.

- Signs of toxicity : no cytotoxicity observed
- Individual plate counts : see attached results
- Mean number of revertant colonies per plate and standard deviation : see attached results

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see attached results
- Negative (solvent/vehicle) historical control data: see attached results

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Concentration Range Finding Test

Range finding Test (Informatory Toxicity Test)
Concentration (µg/plate)	Salmonella typhimurium tester strains
	TA 98				TA 100
	-S9		+ S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	16.7	0.88	21.3	1.02	95.7	1.03	115.0	0.93
DMSO Control	19.0	1.00	23.0	1.00	-	-	108.7	1.00
Ultrapure Water Control	19.0	1.00	21.0	1.00	93.3	1.00	123.3	1.00
5000	18.0	0.95	25.3	1.21	107.3	1.15	129.7	1.05
1600	19.3	1.02	30.7	1.46	100.0	1.07	112.7	0.91
500	20.7	1.09	26.7	1.27	131.3	1.41	122.0	0.99
160	19.3	1.02	22.3	1.06	94.0	1.01	120.0	0.97
50	20.3	1.07	29.7	1.41	102.7	1.10	126.7	1.03
16	19.0	1.00	21.7	1.03	96.0	1.03	120.7	0.98
5	15.7	0.82	29.7	1.41	104.0	1.11	114.7	0.93
NPD (4 µg)	283.3	14.91	-	-	-	-	-	-
SAZ (2 µg)	-	-	-	-	12240	13.11	-	-
2 AA (2 µg)	-	-	1114.7	48.46	-	-	17387	6.00

 MR: Mutation Rate

 Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and the DMSO was applied as vehicle for positive control substances: NPD and 2AA. The mutation rate of the test item, SAZ and untreated control is given referring to the ultrapure water; the mutation rate of NPD and 2AA is given referring to DMSO.

 

Table 2: Summary Table of the Results of the Initial Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentration (µg/plate)	Salmonella typhimurium tester strains																Escherichia coli WP2uvrA
	TA 98				TA 100				TA 1535				TA 1537
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	17.0	0.98	20.3	0.94	88.7	0.81	144.7	1.17	9.0	0.96	11.7	1.09	8.7	1.63	5.3	1.00	22.3	1.24	24.3	1.00
DMSO Control	17.3	1.00	22.3	1.00	-	-	109.7	1.00	-	-	9.0	1.00	7.7	1.00	7.7	1.00	-	-	25.3	1.00
Ultrapure Water Control	17.3	1.00	21.7	1.00	110.0	1.00	123.7	1.00	9.3	1.00	10.7	1.00	5.3	1.00	5.3	1.00	18.0	1.00	24.3	1.00
5000	24.7	1.42	28.7	1.32	105.0	0.95	114.7	0.93	8.3	0.89	13.3	1.25	5.3	1.00	6.7	1.25	23.7	1.31	28.0	1.15
1600	25.7	1.48	29.7	1.37	99.7	0.91	111.0	0.90	10.3	1.11	10.3	0.97	5.7	1.06	7.3	1.38	25.0	1.39	31.0	1.27
500	19.3	1.12	21.0	0.97	105.3	0.96	100.3	0.81	10.7	1.14	10.3	0.97	4.7	0.88	6.0	1.13	21.7	1.20	27.3	1.12
160	20.0	1.15	27.7	1.28	96.0	0.87	91.0	0.74	10.3	1.11	13.3	1.25	6.0	1.13	7.0	1.31	20.3	1.13	24.0	0.99
50	18.0	1.04	17.7	0.82	88.7	0.81	104.3	0.84	10.3	1.11	10.3	0.97	9.0	1.69	5.7	1.06	22.0	1.22	25.0	1.03
16	22.3	1.29	32.7	1.51	91.0	0.83	96.3	0.78	8.7	0.93	11.7	1.09	7.3	1.38	9.7	1.81	16.0	0.89	25.0	1.03
NPD (4 µg)	470.0	27.12	-			-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
SAZ (2 µg)	-	-	-	-	1549.3	-14.08	-	-	979.3	104.93	-	-	-	-	-	-	-	-	-	-
9AA (50 µg)	-	-	-	-	-	-	-	-	-	-	-	-	860.0	112.27	-	-	-	-	-	-
MMS (2 µL)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
2AA (2 µg)	-	-	1176.0	52.66	-	-	1008.0	9.19	-	-	179.3	19.93	-	-	169.0	22.04	-	-	-	-
2AA (50 µg)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	342.0	13.50

 MR: Mutation Rate

Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 3: Historical Control Values for Revertants/Plate (for the Period of 2008-2015)

 

			Bacterial strains
			TA98	TA100	TA1535	TA1537	E. coli
Historical control data of untreated control	-S9	Average	21.4	106.0	10.4	8.1	25.6
		SD	3.7	27.3	1.5	2.5	5.5
		Minimum	9	65	3	2	11
		Maximum	39	157	23	19	45
	+S9	Average	28.0	117.1	11.9	9.0	34.3
		SD	4.2	19.4	1.5	2.0	5.4
		Minimum	12	75	4	3	18
		Maximum	48	166	24	20	56
Historical control data of DMSO control	-S9	Average	20.9	101.4	10.3	7.9	24.9
		SD	3.5	26.2	1.4	2.5	4.9
		Minimum	10	65	3	2	11
		Maximum	39	150	23	20	44
	+S9	Average	27.1	114.7	12.0	8.8	34.2
		SD	4.0	19.3	1.5	2.1	5.2
		Minimum	15	71	4	3	16
		Maximum	48	161	24	20	56
Historical control data of Water control	-S9	Average	22.4	105.5	10.4	7.5	26.3
		SD	3.6	27.6	1.6	2.3	5.9
		Minimum	12	67	3	2	13
		Maximum	36	156	24	15	47
	+S9	Average	28.0	117.4	11.5	8.7	35.2
		SD	4.0	19.8	1.4	2.3	5.2
		Minimum	15	83	4	4	18
		Maximum	43	166	22	16	56
Historical control data of positive controls	-S9	Average	255.6	958.9	842.1	467.4	712.3
		SD	30.7	149.9	134.0	105.7	57.5
		Minimum	123	522	354	109	320
		Maximum	647	1927	1871	1498	1283
	+S9	Average	1224.8	1431.9	165.4	148.0	264.7
		SD	293.8	339.9	35.1	21.3	74.2
		Minimum	409	581	85	68	141
		Maximum	2587	2923	507	407	487

Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2 uvrA

SD: Standard deviation;

DMSO: Dimethyl sulfoxide

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.

Executive summary:

A study according OECD TG 471 was performed with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity.

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:

5000; 1600; 500; 160; 50 and 16 μg/plate.

In the preliminary experiments the pH of the aqueous test item solution (50 mg/mL) was found as 3.47. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests the pH of the test item stock solution (prepared for the highest concentration of 50 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the test item containing overlays was ~7 in both experiments. In this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.